Summary of Core projects active during FY2014: 1. Validation of candidate genes by microinjections of morpholinos and mRNA: 1.1 Assessing neutrophil defect in vps45 morphants using Tg(mpx:GFP) line and assessing migration of neutrophils to the site of injury. 1.2. Zebrafish model of GNE myopathy: An autosomal recessive adult onset muscular dystrophy caused by mutations in the GNE gene. To understand the exact mechanism of muscle atrophy and degeneration, we identified a missense mutation in gne. Gahl lab is currently testing rescue of muscle defects and survival of mutant and morphant embryos and larvae with ManNAC, ManNAz and NeuAc treatment. 1.3 Validation of candidate genes for Joubert Syndrome and related disorders: JSRDs are a clinically and genetically heterogenous group of ciliopathies defined based on a distinctive malformation (molar tooth sign) on brain imaging. To date, 21 genes have been identified, but they account for only 50% of JSRDs. To identify candidate genes in remaining 50% of JSRDs, the Gahl lab has evaluated 103 JSRD patients from 89 families. Whole exome sequencing identified 5 novel candidate genes. Zebrafish model is being used to ascertain their role in primary cilium, basal body, or centrosome biogenesis and function. Currently, we are investigating the role of an uncharacterized gene kiaa0753 using morpholinos and CRISPR/Cas9 nuclease-mediated targeted knockout mutants. Preliminary data with two morpholinos and WISH using markers of cilium function is consistent with role of kiaa0753 in cilium biogenesis. 1.4 Validation of TRNT1 as the causative gene for a new syndrome: Mutations in TRNT1 were identified in patients from 4 unrelated families with a new syndrome characterized by recurrent fever, severe anemia, and immunologic, neurologic and gastrointestinal symptoms. Zebrafish embryos injected with translation-blocking and splice blocking morpholinos showed reduced hemoglobin staining (anemia) in addition to other morphological phenotypes. To prove specificity of the morpholino phenotype, we performed complementation experiments with human wildtype and mutant TRNT1 mRNAs. 1.5 Evaluation of candidate genes for UDP group: The UDP group identifies candidate genes in patients with rare diseases and zebrafish modeling is used to validate their role in the disease phenotype. We are currently analyzing sccpdha using morpholino knockdown strategy to understand its role in brain development. Dr. Burke is applying WISH, histology, and microarrays to characterize the phenotypes associated with loss and gain of functions of sccpdha. Several rounds of injections of the sccpdha morpholinos have been performed for these experiments. 2. Generation of knockout mutants using targeted mutagenesis: In collaboration with the Genomics Core, we developed high throughput method of fluorescent PCR for founder screening and genotyping to complete these projects in a cost-effective and timely fashion. A brief summary of the projects is given below: 2.1 Role of AK2 in reticular dysgenesis: To model reticular dysgenesis in zebrafish and investigate the mechanisms underlying its pathophysiology, we generated multiple ak2 mutants by TILLING and ZFNs. Dr. Rissone has performed a comprehensive study of the effects of ak2 deficiency on hematopoiesis using these mutants. His data showed that ak2 plays a critical role in erythroid development during primitive hematopoiesis and hematopoietic stem cell (HSC) development during definitive hematopoiesis. Mutants were also used to test antioxidants as possible therapeutics by rescuing the phenotype. 2.2 Regulation of hematopoiesis: a. SOX17: To investigate the role of sox17 in the transition of hemogenic endothelium into nascent hematopoietic stem cells, we generated two loss-of-function alleles using ZFNs. Liu lab is currently analyzing expression of specific hematopoietic markers by WISH and transgenic lines.. b. RUNX family of genes: Proteins encoded by RUNX family of genes (runx1, runx2a, runx2b and runx3) heterodimerize with CBF&#946; and play critical roles during definitive hematopoiesis, bone development and other biological processes. Liu lab is characterizing the runx1 knockout mutants generated by TALENs (assembled using golden gate kit in the core) to understand the source of HSCs for adult hematopoiesis. We are helping Dr. Bresciani to generate mutants in runx2a, runx2b and runx3 using CRISPR/Cas9 nucleases. 2.3 Modeling metabolic diseases in zebrafish: a. MMACHC: Cobalamin C (cblC) disease is a rare inborn error of vitamin B12 metabolism, for which there is no animal model. To model cblC disease for insights into the underlying pathology and assessment of the efficacy of potential therapeutics, we generated two loss-of-function alleles in mmachc using ZFNs. Dr. Sloan and Mr. Achilly performed detailed functional studies analyzing growth parameters, drug sensitivity, survival, gene expression changes using qPCR and microarrays, cell composition studies in the blood and retina using flow cytometry, histology, metabolite values, and responses to conventional and novel therapeutics. Their results demonstrated that the mmachc mutant fish model successfully recapitulates the human phenotype, thereby allowing us to better understand the pathophysiology and explore potential therapeutics. Future directions include RNA-seq, studies of rod and cone photoreceptors, electron microscopy of the retina, ERG studies, and additional drug screens. b. MUT: We generated mutant fish by ZFNs for mut methylmalonic academia, a disorder of vitamin B12 metabolism. The homozygous mutant fish die between 7-14 dpf. Venditti lab is analyzing them by histology and gene expression. 2.4 Zebrafish models of new candidate genes from Gahl lab: a. AIFM2: Identified as a candidate gene in a family with a new immune disorder. We generated 2 mutant alleles using TALENs obtained through the Center for Regenerative Medicine grant from Cellectis. We will help Dr. Vilboux to analyze their neutrophil compartment using response to injury and bacterial infection assays. b. PUS3: Identified as a candidate gene in a family with Kabuki-like syndrome, a disorder characterized by distinctive facial features, similar to traditional Japanese theater called Kabuki. Patients also display neurological and/or muscular defects. TALENs from Cellectis did not show any activity by somatic analysis of the injected embryos. Therefore, we targeted pus3 with CRISPRs and are in the process of phenotype analysis of the mutant embryos. c. KIAA0753: First of the 5 candidate genes identified for ciliopathy disorders (JSRDs): In addition to evaluating the embryonic phenotype by morpholinos, we are generating mutant lines with CRISPRs to evaluate the larval and adult phenotypes. 4. Generation of stable transgenic lines: We generated stable transgenic lines for two constructs: GFP tagged riboprotein for mRNA immunoprecipitation and HyPerYFP to detect reactive oxidative species generation using Tol2 mRNA and plasmid DNA. We also used Zebrafish as in vivo system to test conserved regulatory sequences as enhancers by tol2 transgenesis and a minimum promoter plus the fragment to be tested driving GFP. 5. Chemical screening: The Core is facilitating characterization of the role of elg1 in DNA repair by testing sensitivity of elg1 mutant fish to various DNA damaging agents. Several potential therapeutic chemicals were tested by Candotti, Venditti and Gahl labs on the mutants and morphants in their genes of interest. 6. Cryopreservation: The Core extracted testes from at least two males/line from more than 50 mutant lines generated by the Core, Liu lab and Burgess labs for cryopreservation. In addition, the Core provided training for performing cryopreservation to users.